Integrin activation is required for platelet aggregation in thrombosis and hemostasis and plays an important role in the migration of endothelial cells and in angiogenesis. Our previous work led to the discovery of an Integrin Activation Complex (IAC) containing Rap-interacting Adaptor Molecule (RIAM), talin, and integrins. To test the hypothesis that the IAC contains a characteristic population of proteins, we will characterize the IAC in detail by establishing its protein composition by use of a novel tandem affinity tag approach to purify unoccupied integrins in complex with RIAM and utilize mass spectrometry to specify the protein cartography (proteome) of these isolated complexes. Bioinformatics will be used to project the identified proteome onto physical, genetic, and pathway protein-protein interaction networks to help select identified IAC components for validation by direct immunoprecipitation and purified protein-protein interactions. To test the hypothesis to that IACs of differing composition exist in specific cellular compartments, we will devise a novel bimolecular fluorescence complementation method to visualize the IAC and relate its position to specific cellular locales e.g. lamellipodium or specific vesicle populations. To test the hypothesi to that components of the IAC have specific roles in the formation or behavior of the IAC and in integrin activation and cell migration, we will examine the effects of elimination of IAC components on the movements of the IAC, integrin activation, cell migration, actin flow, and protrusion morphodynamics. These studies will establish biochemical and cell biological means to characterize and study modular assemblies of proteins that control integrin activation and the integrin-dependent regulation of actin dynamics and cell migration. The work will therefore provide new insights into the mechanisms of integrin activation involved in arterial thrombosis and inflammation. The studies will also contribute insights into mechanisms controlling the migration of vascular cells.